The destruction of tissue in inflammation caused by nonimmunological and immunological processes induces the formation of different endogenic substances (mediators and hormones). They regulate the complex steps of activation of the inflammation and tissue regeneration processes. The mediators are formed either by limited and regulated proteolysis of plasma and serum protein factors as humoral mediators; or they are liberated by active secretion and/or cell lysis from cells and tissues as cellular mediators. The mediators and hormones are especially important as specific carriers of chemical information which are formed and secreted by leukocytes in the course of cell proliferation processes (mitosis processes). They are components of the body's defence system whose systemic and local activation they regulate. The mediators contribute to the removal and detoxification of the body's own components and/or foreign components. In addition, by regulation of cell proliferation and tissue growth processes in wound-healing, they contribute to the restoration of physiological functions of the organism. As the classical hormones of endocrine glands, inflammatory mediators are trace components of tissues or blood and are present in very minute concentrations only. Experimental evidence shows that only up to 5,000 of such mediator protein molecules can be maintained in a steady state equilibrium by a cell in the mitotic cycle in its surrounding medium.
A reaction resulting in the division of a cell accompanied by doubling of the chromosome set is called "mitosis". Leukocytes comprise the mature, fully differentiated circulating type of granulocytes (segmented neutrophilic, eosinophilic and basophilic phagocytes) and the mononuclear leukocytes (monocytic phagocytes and lymphocytes) with their different sub-species (T, B-cells, etc.). Of these circulating cells, only the mature mononuclear leukocytes are capable of further division and differentiation. To the precursors of these mature leukocyte types for the development and maturation line (hematopoiesis) of the segmented granulocytes (Leukopoiesis) belong in particular the granulocytic bands. Their precursor is the mature and juvenile metamyelocyte which develops from the sequence of myelocyte, promyelocyte, myeloblast and bone marrow stem cell.
The body's own endogenous chemical substances which control and regulate the processes of this cell defferentiation and maturation line are called leukopoietins; see H. E. Whipple and M. I. Spitzer (eds.), "Leukopoiesis in Health and Disease, Ann. N.Y. Acad. Sci., 113 (1964), p. 511 to 1092. The leukopoietins include, amongst others, substances which induce the division and differentiation of the leukocytes and of their precursors (mitogens).
There exist numerous publications on the mitosis of peripheral and tissue mononuclear leukocytes and of leukocyte precursors of the bone marrow which are capable of division; see J. Lobue and S. A. Gordon, "Humoral Control of Growth and Differentiation", vol. I, Academic Press, New York, 1973. There also exist reports on different types of biological activities, for instance in cell cultures, with mitogenic effects on leukocytes or their precursors. Such an activity is for instance the "colony-stimulating factor" described by T. R. Bradley and D. Metcalf, Aust. J. Exp. Biol. Med. Sci. 44 (1966), p. 287 as well as by numerous other authors. This term comprises all activities which can, for instance, be observed in serum or urine extracts. This factor is capable of stimulating the proliferation and differentiation of granulocytes and macrophage precursors in in vitro cultures.
A similar activity is the "T cell growth factor" described by D. Y. Mochizuki et al. J. Immunol. Meth. 39 (1980), p. 185 to 201. This activity is said to originate for instance from T-cells and macrophages and is capable of maintaining T-cell clones in long-lasting cultures in vitro.
The designation LAF (lymphocyte-activating factor) applies to another biological activity described; see I. Gery and R. E. Handschuhmacher, Zell. Immunol. 11 (1974), p. 162.
To date, it has not been possible to isolate and define any of the numerous mitosis-stimulating activities as specified substance. On the contrary, all papers only deal with the experimental proof of the mitosis-stimulating activity of chemically undefined solutions or mixtures. Therefore, there is no certain knowledge on the chemical nature of the active substances and their biological specifity.
The mitosis activity is measured as "mitosis-index" of a cell population. The ratio of the number of cells in mitotic cycle to the overall number is measured by chromosome analysis. Another test is based on the incorporation of radioactive thymidine; see J. Paul, "Cell and Tissue Cultures" 5th edition, 1975, Churchill Livingstone, London.
It is therefore a primary object of this invention to provide a new class of cellular mitogens from leukocytes.
It is another object of this invention to provide a new class of cellular mitogens from leukocytes in highly purified form.
It is another of this invention to provide a new class of cellular mitogens from leukocytes in physical quantities for practical use.
It is another object of this invention to provide a new class of mitogens from leukocytes, which represent biologically specific, active and naturally acting mediators stimulating cell division and differentiation of leukocytes and/or their precursors.
It is another object of this invention to provide a new class of mitogens from leukocytes, which are suitable for specifically influencing the defense state of mammalian (e.g. human) organisms.
It is still another object of this invention to provide a process for producing and obtaining a new class of mitogens from leukocytes in an economical, biotechnically useful and relatively simple manner.
It is still another object of this invention to provide a process for producing and obtaining a new class of mitogens from leukocytes in a highly purified, molecularly homogeneous form and in physical quantities for practical use.
It is still another object of this invention to provide a pharmaceutical composition for specifically influencing the defence state of the body of mammalians.
These and other objects and advantages of the present invention will be evident from the following description of invention.